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Sep 2017

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Quantitative Live-cell Reporter Assay for Noncanonical Wnt Activity
可定量的活细胞报告基因用于检测非经典Wnt活动   

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Abstract

Noncanonical Wnt signaling functions independently of the β-catenin pathway to control diverse developmental processes, and dysfunction of the pathway contributes to a number of human pathological conditions, including birth defects and metastatic cancer. Progress in the field, however, has been hampered by the scarcity of functional assays for measuring noncanonical Wnt signaling activity. We recently described the Wnt5a-Ror-Kif26b (WRK) reporter assay, which directly monitors a post-transcriptional regulatory event in noncanonical Wnt signaling. In this protocol, we describe the generation of the stable GFP-Kif26b reporter cell line and a quantitative reporter assay for detecting and measuring Wnt5a signaling activities in live cells via flow cytometry.

Keywords: Noncanonical Wnt reporter (非经典Wnt报告基因), Wnt5a signaling (Wnt5a信号转导), Kif26b (Kif26b), Regulated degradation (受调控的降解), Flow cytometry (流式细胞术)

Background

Historically, transcriptional reporter assays have facilitated the delineation of major signaling pathways. In particular, β-catenin-dependent luciferase- or GFP-based transcriptional reporters have been instrumental in elucidating the molecular mechanisms of the canonical Wnt/β-catenin pathway (Korinek et al., 1997; Fuerer and Nusse, 2010). Although a number of noncanonical Wnt signaling reporters based on JNK-dependent transcription have been described, it remains unclear whether these transcriptional responses are primary or secondary to noncanonical Wnt signaling (Veeman et al., 2003; Nishita et al., 2010; Ohkawara and Niehrs, 2011). Also, a reporter for real-time detection of non-transcriptional Wnt5a-Ror signaling events has not been available. The Wnt5a-Ror-Kif26b (WRK) reporter assay, which directly monitors a non-transcriptional Wnt5a-Ror signaling event, adds to the current repertoire of molecular tools for studying noncanonical Wnt signaling (Ho et al., 2012; Susman et al., 2017).

As described in our recent publication, Wnt5a-Ror signaling modulates the steady-state protein level of the kinesin superfamily member Kif26b by inducing its ubiquitin- and proteasome-dependent degradation (Susman et al., 2017). This reporter assay enables further identification and mechanism-based analysis of other Wnt5a-Ror signaling components, most of which remain unknown or relatively unexplored. In addition, the WRK assay may also facilitate the screening of pharmacological agents in Wnt5a-Ror related diseases such as certain cancers and developmental disorders.

This protocol describes the generation of the stable GFP-Kif26b reporter cell line and a quantitative method of detecting Wnt5a signaling levels in live GFP-Kif26b reporter cells via flow cytometry.

Materials and Reagents

  1. Pipette tips (USA Scientific, catalog numbers: 1122-1832 , 1120-8812 , 1123-1812 , 1121-3812 )
  2. 10-cm tissue culture dish (Corning, Falcon®, catalog number: 353003 )
  3. 1.5 ml microcentrifuge tubes (Denville Scientific, catalog number: C2170 )
    Note: Autoclave before use.
  4. 6-well plate
  5. 48-well tissue culture plate (Corning, Costar®, catalog number: 3548 )
  6. 5 ml round-bottom tubes with 35 µm cell strainer snap cap (Corning, Falcon®, catalog number: 352235 )
  7. NIH/3T3 Flp-In cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: R76107 )
  8. pCAG-GFP (available upon request), or any GFP plasmid suitable for mammalian expression
  9. pEF5-FRT-GFP-Kif26b (Addgene, catalog number: 102862 ) reporter construct
  10. pOG44 Flp-Recombinase expression vector (Thermo Fisher Scientific, InvitrogenTM, catalog number: V600520 )
  11. Recombinant Wnt5a (R&D Systems, catalog number: 654-WN-010 )
  12. Genjet In Vitro Transfection Reagent for NIH/3T3 cells (SignaGen Laboratories, catalog number: SL100488 , 3T3)
  13. Hygromycin B (50 mg/ml solution) (Corning, Mediatech, catalog number: 30-240-CR )
  14. Poly-D-lysine (Sigma-Aldrich, catalog number: P6407-10X5MG )
  15. Wnt-C59 (Cellagen Technology, catalog number: C7641-2s )
  16. Trypsin EDTA (Corning, Mediatech, catalog number: 25-052-CI )
  17. Dulbecco’s modified Eagle’s medium (DMEM) (Corning, Mediatech, catalog number: 15-017-CV )
  18. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16000069 )
    Note: The FBS is used directly without heat-inactivation.
  19. Glutamine  (100x solution, 200 mM) (Corning, Mediatech, catalog number: 25-005-CI )
  20. Penicillin-streptomycin (100x solution, 100 IU/ml) (Corning, Mediatech, catalog number: 30-002-CI )
  21. Bovine serum albumin (BSA) (Fisher Scientific, catalog number: BP1600-1 )
  22. CHAPS detergent (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28300 )
  23. Phosphate buffered saline (PBS) (GE Healthcare, catalog number: SH30256.01 )
  24. Growth media (see Recipes)
  25. Wnt control buffer (see Recipes)
  26. Cell resuspension buffer for flow cytometry (see Recipes)

Equipment

  1. Pipetters (e.g., Eppendorf, model: Research® plus )
  2. 37 °C, 5% CO2 incubator (e.g., Heracell by Thermo Fisher Scientific)
  3. Centrifuge with cooling capabilities (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM LegendTM Micro 21R )
  4. Fluorescent microscope with 488 nm light source (e.g., Thermo Fisher Scientific, model: EVOS® )
  5. Flow cytometer with 488 nm laser (e.g., BD, model: FACScan )

Software

  1. FlowJo software (FlowJo, LLC; https://www.flowjo.com/)

Procedure

  1. Generation of stable reporter cell lines using the Flp-In NIH/3T3 cell line
    1. Cell plating for transfection
      Seed cells at 1.62 M cells/plate in a 10-cm plate in 10 ml of growth media. Culture the cells at 37 °C until they reach 80% confluency (about 18-24 h, Figure 1).
      Note: Prepare 1 plate of cells for each reporter construct, plus 1 additional plate for the pCAG-GFP, which serves as both a negative control for the Flp-In and a reference for transfection efficiency.


      Figure 1. Confluency (80%) at the time of transfection. Phase contrast, 10x magnification. Scale bar represents 400 μm.

    2. Transfection
      1. One hour before transfection, remove media from cells and replace with 6 ml fresh growth media.
      2. Dilute DNA: In a 1.5 ml microcentrifuge tube, add 1.35 μg pEF5-FRT-GFP-Kif26b and 12.15 μg pOG44 to 675 μl of serum-free media (plain DMEM). In parallel, for the GFP control plate, prepare a tube of 675 μl serum-free media with 13.5 μg of pCAG-GFP but no pOG44. Mix well by pipetting.
        Note: Total mass of transfected DNA is 13.5 μg. Transfect with a 1:10 molar ratio of reporter plasmid to flp recombinase; adjust masses according to the size of the plasmid.
      3. Dilute the GenJet transfection reagent: for each plate, prepare a separate 1.5 ml microcentrifuge tube of 40.5 μl GenJet transfection reagent in 675 μl of serum-free media (plain DMEM). Mix well by pipetting.
      4. Add each tube of diluted GenJet solution all at once to each respective DNA solution.
        Note: The GenJet solution must be added to the DNA solution, not the reverse. Vortex gently for 4 sec to mix.
      5. Incubate the transfection mixes for 15 min at room temperature. Do not let the incubation proceed for more than 20 min.
      6. Add the transfection mixes drop-wise to their respective plates of cells.
      7. Gently rock the plates to mix well and return the plates to the incubator.
      8. After 12-18 h, check transfection efficiency by visualizing the GFP control plate under a fluorescent microscope (Figure 2). Remove transfection media and replace with 10 ml of growth media.


        Figure 2. GFP control plate 12-18 h after transfection. A. Phase contrast channel, 10x magnification. Scale bar represents 400 μm. B. GFP channel, 10x magnification. Scale bar represents 400 μm.

    3. Antibiotic selection
      1. Two days after transfection, split each 10-cm plate into 4 x 10-cm plates in growth media to avoid overcrowding cells during selection (do not use selection antibiotics during the split).
      2. After cells adhere to the plate, remove media and replace with fresh growth media containing 200 μg/ml hygromycin B. Replace with fresh hygromycin media every 3-4 days. Selection should take about 7-10 days. Between 6-20 colonies per plate is typically expected (Figure 3).
        Note: A kill curve was conducted to determine that 200 μg/ml hygromycin B is optimal for NIH/3T3 Flp-In cells. The optimal selection concentration may vary slightly depending on the source of hygromycin B and cell lines.


        Figure 3. A representative colony at 7 days post-hygromycin B selection. Phase contrast, 4x magnification. Scale bar represents 1,000 μm.

      3. Cells may be pooled from 1 or 2 10-cm plates into a single well of a 6-well plate and passaged in growth media without selection antibiotics.
        Note: This step is only performed for the reporter constructs. The GFP control plate, which should yield no colonies, is discarded.

  2. Wnt5a stimulation assay
    Experimental design: For a basic Wnt5a stimulation, include one condition for stimulation (+Wnt5a, where Wnt5a-containing media is added) and one condition for control (-Wnt5a, where control buffer-containing media is added) for each reporter cell line. The experiment setup will vary depending on your application of the assay; see Data analysis section for details on other types of stimulations.
    1. Seed reporter cells at 0.09 million/well in the poly-D-lysine-coated 48-well plate in 400 μl growth media per well. Cells should be about 90% confluent.
      Notes:
      1. Plate coating is done by adding 200 μl of a poly-D-lysine solution (0.1 mg/ml in water; sterile filtered) to each well of a 48-well plate, incubating at room temperature for 15 min, removing the poly-D-lysine solution, and washing the wells with 400 μl of water three times. Air dry the plate completely (with the lid removed) before plating cells. Coated plates can also be stored at room temperature for future use.
      2. For quantification, we typically plate cells in triplicate wells for each experimental condition.
    2. The next day, gently remove media and replace with 400 μl growth media containing 10 nM Wnt-C59. Wnt-C59 inhibits the processing and secretion of endogenous Wnts. Allow cells to reach 100% confluency in Wnt-C59-containing media (generally one day). Cells should be as confluent as possible on the day of Wnt5a stimulation.
      Note: If the monolayer of cells retract or peel off, repeat cell plating. Retracted cells do not signal well.
    3. To stimulate cells with Wnt5a, gently remove media and replace with media containing 10 nM Wnt-C59 and the respective concentration of Wnt5a. For mock stimulation, use media containing Wnt-C59 and Wnt control buffer. If other drugs are used in conjunction with Wnt5a, pretreatment of the drug (typically for 1 h) may be necessary before addition of Wnt5a- and drug-containing media. Avoid disturbing the cell monolayer during media change.
    4. Incubate cells with Wnt5a at 37 °C for 6 h.
    5. To harvest cells for flow cytometry analysis, dissociate the cells with 100 μl trypsin per well at 37 °C for 3-5 min. Neutralize the trypsin with 500 μl of growth media and transfer the cell suspensions to 1.5 ml microcentrifuge tubes.
    6. Centrifuge cells at 12,000 x g at 4 °C for 3 min to pellet the cells.
    7. Remove the supernatant from each sample. Avoid disturbing the pellet.
    8. Resuspend the pellets at room temperature in 100-150 μl flow cytometer buffer. Mix by pipetting until the sample is homogenously resuspended and strain the cell suspension into a round-bottom tube through the strainer cap.
    9. Analyze the cells using a flow cytometer. We routinely use the Becton Dickinson FACScan and analyze 30,000 cells per sample.
    10. Analyze data files in software (e.g., FlowJo). See next section for details.

Data analysis

  1. For general data analysis, gate the live cell population via side scatter and forward scatter parameters in the flow cytometry software to exclude dead cells. The wild-type NIH/3T3 Flp-In parent cell line (i.e., untransfected) is used as a reference for autofluorescence; however, we do not typically gate the cell population based on the GFP signal to ensure that the entire live cell population is included in the reporter analysis. Generate a raw histogram of GFP fluorescence vs. cell count for the live gated population. Overlay the histograms from each sample to be compared to obtain the difference in median fluorescence between each sample population (Figure 4). This difference in medians is expressed as a percentage: [(Control median - stimulated median)/control median] x 100 (labeled as ‘% downregulation’ in Figure 5B). Multiple histograms may be overlaid for comparison or reference (Figure 5A, Figure 7A).


    Figure 4. Basic analysis using the WRK reporter assay. Overlaid histograms from one set of samples showing the downregulation of GFP-Kif26b fluorescence in the WRK reporter cell line after Wnt5a stimulation (0.2 μg/ml Wnt5a) for 6 h.

  2. For a dose-response analysis, we analyze a minimum of six samples with varying concentrations of the Wnt5a ligand or small molecule inhibitors, including a 0 dose point. The medians may be plotted against the concentrations to generate the dose-response curve (Figure 5B). For inhibitors, we typically vary the drug concentration in the presence of a fixed concentration of Wnt5a to determine the dose-response relationship.


    Figure 5. Example of a dose-response analysis using the WRK reporter assay. Raw histograms (A) and the resulting dose-response curve (B) showing GFP-Kif26b downregulation as a function of Wnt5a concentration in the WRK reporter assay.

  3. For a time course experiment, such as the Kif26b stability analysis shown in Figure 6, we stimulate samples with Wnt5a at regular time intervals until the end of the experiment, when all samples are harvested at once. The medians are plotted against the duration of stimulation.


    Figure 6. Example of a time course experiment using the WRK reporter assay. The kinetics of GFP-Kif26b turnover in the absence or presence of Wnt5a stimulation, as measured in the WRK reporter assay. Cycloheximide was used to block new protein synthesis in the reporter cells.

  4. For statistical analysis during quantification, we use a minimum of three biological replicates (cells plated and treated with Wnt5a and/or inhibitors in concurrent cultures). To assess the difference between two sets of data, we perform a two-tailed, unpaired Student’s t-test (Figure 7B). We include error bars for each set of replicates representing the standard error of the mean, which we generate by calculating the standard deviation of the medians of the replicates and dividing that number by the square root of N, where N is the number of replicates (Figure 6, Figure 7B).


    Figure 7. Example of a pathway analysis experiment using the WRK reporter assay. Partial blocking of Wnt5a-induced reporter activity after ectopic Shisa2 expression via lentiviral transduction. A. Representative overlaid histograms show the effect of ectopic Shisa2 expression on Wnt5a-induced downregulation of GFP-Kif26b in the WRK reporter line. Shisa2 is an antagonist of the Frizzled family of Wnt receptor (Yamamoto et al., 2005). The effect of Wnt5a or control buffer treatment on the WRK reporter line is included as a reference. B. Quantification of the results shown in panel (A). t-tests were performed for the following comparisons: Control virus vs. no virus, P = 0.0957 (not significant); control virus vs. Shisa2 virus, P < 0.001 (significant).

Notes

Wnt5a signaling as detected by this assay appears to be highly sensitive to cell density. Signaling activity occurs best when cells are as confluent as possible, and activity decreases drastically when cells are less than 100% confluent. Some optimization may be required to determine the most optimal plating conditions for specific cell types and applications.

Recipes

  1. Growth medium
    DMEM supplemented with:
    10% FBS
    1x glutamine (2 mM)
    1x penicillin-streptomycin (1 IU/ml)
  2. Wnt control buffer
    1x PBS supplemented with:
    0.1% bovine serum albumin
    0.5% (w/v) CHAPS
  3. Cell resuspension buffer for flow cytometry
    1x PBS supplemented with 0.5% FBS

Acknowledgments

This protocol was adapted from the following paper: Susman et al., 2017. We thank Michael Greenberg (Harvard Medical School) for discussions and support. We thank Bridgette McLaughlin at the UC Davis Cancer Center Flow Cytometry core (supported by P30 CA093373) for providing instruments, training and support. The development and characterization of the WRK reporter assay was supported by American Cancer Society grant IRG-95-125-13 and National Institutes of Health grant 1R35GM119574-01 to H.H. Ho, and T32GM007753 to M.W. Susman. The authors declare no conflicts of interest.

References

  1. Fuerer, C. and Nusse, R. (2010). Lentiviral vectors to probe and manipulate the Wnt signaling pathway. PLoS One 5(2): e9370.
  2. Ho, H. Y., Susman, M. W., Bikoff, J. B., Ryu, Y. K., Jonas, A. M., Hu, L., Kuruvilla, R. and Greenberg, M. E. (2012). Wnt5a-Ror-Dishevelled signaling constitutes a core developmental pathway that controls tissue morphogenesis. Proc Natl Acad Sci U S A 109(11): 4044-4051.
  3. Korinek, V., Barker, N., Morin, P. J., van Wichen, D., de Weger, R., Kinzler, K. W., Vogelstein, B. and Clevers, H. (1997). Constitutive transcriptional activation by a β-catenin-Tcf complex in APC-/- colon carcinoma. Science 275(5307): 1784-1787.
  4. Nishita, M., Itsukushima, S., Nomachi, A., Endo, M., Wang, Z., Inaba, D., Qiao, S., Takada, S., Kikuchi, A. and Minami, Y. (2010). Ror2/Frizzled complex mediates Wnt5a-induced AP-1 activation by regulating Dishevelled polymerization. Mol Cell Biol 30(14): 3610-3619.
  5. Ohkawara, B. and Niehrs, C. (2011). An ATF2-based luciferase reporter to monitor non-canonical Wnt signaling in Xenopus embryos. Dev Dyn 240(1): 188-194.
  6. Susman, M. W., Karuna, E. P., Kunz, R. C., Gujral, T. S., Cantu, A. V., Choi, S. S., Jong, B. Y., Okada, K., Scales, M. K., Hum, J., Hu, L. S., Kirschner, M. W., Nishinakamura, R., Yamada, S., Laird, D. J., Jao, L. E., Gygi, S. P., Greenberg, M. E. and Ho, H. H. (2017). Kinesin superfamily protein Kif26b links Wnt5a-Ror signaling to the control of cell and tissue behaviors in vertebrates. Elife 6.
  7. Veeman, M. T., Axelrod, J. D. and Moon, R. T. (2003). A second canon. Functions and mechanisms of β-catenin-independent Wnt signaling. Dev Cell 5(3): 367-377.
  8. Yamamoto, A., Nagano, T., Takeara, S., Hibi, M. and Aizawa, S. (2005). Shisa promotes head formation through the inhibition of receptor protein maturation for the caudalizing factors, Wnt and FGF. Cell 120(2): 223-35.

简介

非经典Wnt信号独立于β-catenin途径发挥功能来控制不同的发育过程,并且该途径的功能障碍导致许多人类病理状况,包括出生缺陷和转移性癌症。 然而,该领域的进展一直受到用于测量非典型Wnt信号活性的功能测定的稀缺性的阻碍。 我们最近描述了Wnt5a-Ror-Kif26b(WRK)记者测定法,其直接监测非典型Wnt信号传导中的转录后调节事件。 在该协议中,我们描述了通过流式细胞术检测和测量活细胞中Wnt5a信号传导活性的稳定GFP-Kif26b报告细胞系和定量记者测定法的产生。

【背景】从历史上看,转录报告基因检测方法促进了主要信号通路的描述。具体来说,β-连环蛋白依赖性萤光素酶或基于GFP的转录报道基因有助于阐明经典Wnt /β-连环蛋白途径的分子机制(Korinek et al。,1997; Fuerer and Nusse,2010)。尽管已经描述了许多基于JNK依赖性转录的非经典Wnt信号传导报道分子,但是这些转录应答是否是非经典Wnt信号传导的主要或次要仍不清楚(Veeman等,2003; Nishita等,等,2010; Ohkawara和Niehrs,2011)。此外,用于实时检测非转录性Wnt5a-Ror信号传导事件的记者尚未获得。 Wnt5a-Ror-Kif26b(WRK)记者测定法直接监测非转录性Wnt5a-Ror信号事件,增加了研究非经典Wnt信号传导的分子工具的最新成分(Ho等人 ,2012; Susman等人,2017)。

如我们最近的公开文献中所述,Wnt5a-Ror信号通过诱导驱动蛋白超家族成员Kif26b的泛素和蛋白酶体依赖性降解来调节稳态蛋白水平(Susman等人,2017)。本报告分析能够进一步鉴定其他Wnt5a-Ror信号成分,并基于机制分析,其中大部分Wnt5a-Ror信号成分保持未知或相对未开发。另外,WRK测定还可以有助于在Wnt5a-Ror相关疾病如某些癌症和发育障碍中筛选药理学试剂。

该协议描述了稳定的GFP-Kif26b报告细胞系的产生以及通过流式细胞术检测活GFP-Kif26b报道细胞中Wnt5a信号水平的定量方法。

关键字:非经典Wnt报告基因, Wnt5a信号转导, Kif26b, 受调控的降解, 流式细胞术

材料和试剂

  1. 移液器吸头(USA Scientific,产品目录号:1122-1832,1120-8812,1123- 1812,1121-3812)
  2. 10厘米组织培养皿(Corning,Falcon ,产品目录号:353003)
  3. 1.5ml微量离心管(Denville Scientific,目录号:C2170)
    注意:在使用前进行高压灭菌。
  4. 6孔板
  5. 48孔组织培养板(Corning,Costar ,目录号:3548)
  6. 5毫升带35微米细胞过滤器卡帽的圆底管(Corning,Falcon ,产品目录号:352235)
  7. NIH / 3T3 Flp-In细胞(Thermo Fisher Scientific,Invitrogen TM,目录号:R76107)
  8. pCAG-GFP(可根据要求提供)或适用于哺乳动物表达的任何GFP质粒
  9. pEF5-GFP-FRT-Kif26b(Addgene,目录号:102862)记者构建物
  10. pOG44 Flp-重组酶表达载体(Thermo Fisher Scientific,Invitrogen TM,目录号:V600520)
  11. 重组Wnt5a(R&amp; D Systems,目录号:654-WN-010)
  12. Genjet体外用于NIH / 3T3细胞的转染试剂(SignaGen Laboratories,目录号:SL100488,3T3)
  13. 潮霉素B(50mg / ml溶液)(Corning,Mediatech,目录号:30-240-CR)
  14. 聚-D-赖氨酸(Sigma-Aldrich,目录号:P6407-10X5MG)
  15. Wnt-C59(Cellagen Technology,目录号:C7641-2s)
  16. 胰蛋白酶EDTA(Corning,Mediatech,目录号:25-052-CI)
  17. 达尔伯克改良伊格尔培养基(DMEM)(Corning,Mediatech,目录号:15-017-CV)
  18. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:16000069)
    注意:FBS直接使用时不需加热灭活。
  19. 谷氨酰胺(Corning,Mediatech,目录号:25-005-CI)
  20. 牛血清白蛋白(BSA)(Fisher Scientific,目录号:BP1600-1)
  21. CHAPS清洁剂(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:28300)
  22. 磷酸盐缓冲盐水(PBS)(GE Healthcare,目录号:SH30256.01)
  23. 生长介质(见食谱)
  24. Wnt控制缓冲区(请参阅食谱)
  25. 用于流式细胞术的细胞重悬缓冲液(参见食谱)

设备

  1. 移液器(例如,Eppendorf,型号:Research ® plus)
  2. 37℃,5%CO 2培养箱(例如,Thermo Fisher Scientific的Heracell)。
  3. 具有冷却能力的离心机(例如,Thermo Fisher Scientific,Thermo Scientific TM TM,型号:Sorvall TM Legend TM TM Micro 21R)
  4. 带有488nm光源的荧光显微镜(例如,Thermo Fisher Scientific,型号:EVOS )
  5. 具有488nm激光的流式细胞仪(例如,BD,型号:FACScan)

软件

  1. FlowJo软件(FlowJo,LLC; https://www.flowjo.com/ )

程序

  1. 使用Flp-In NIH / 3T3细胞系产生稳定的报道细胞系
    1. 用于转染的细胞电镀
      在10ml生长培养基中的10-cm平板中以1.62M细胞/板的种子细胞。
      在37°C培养细胞,直到达到80%汇合(约18-24小时,图1)。
      注意:为每个报告基因构建物准备1个细胞板,加上另外1个pCAG-GFP板,该板用作Flp-In的阴性对照和转染效率的参考。 />

      图1.转染时汇合度(80%)。 相位对比,10倍放大。比例尺表示400微米。

    2. 转染
      1. 转染前一小时,从细胞中取出培养基并用6毫升新鲜生长培养基替代。
      2. 稀释DNA:在1.5ml微量离心管中,向675μl无血清培养基(普通DMEM)中加入1.35μgpEF5-GFP-FRT-Kif26b和12.15μgpOG44。平行地,对于GFP对照板,准备含有13.5μgpCAG-GFP但不含pOG44的675μl无血清培养基的管。通过移液充分混合。
        注:转染DNA的总质量为13.5μg。用报道质粒与flp重组酶的1:10摩尔比例转染;根据质粒的大小调整质量。
      3. 稀释GenJet转染试剂:对于每个平板,在675μl无血清培养基(普通DMEM)中制备40.5μlGenJet转染试剂的单独1.5 ml微量离心管。通过移液很好地混合。
      4. 一次性将每管稀释的GenJet溶液添加到各自的DNA溶液中。
        注意:GenJet解决方案必须添加到DNA溶液中,而不是相反。轻轻振荡4秒钟混合。
      5. 在室温下孵育转染混合物15分钟。不要让孵化进行超过20分钟。
      6. 将转染混合物逐滴添加到各自的细胞培养板中。
      7. 轻轻摇动盘子以充分混合并将盘子送回培养箱。
      8. 12-18小时后,通过在荧光显微镜下观察GFP对照板来检查转染效率(图2)。去除转染培养基,并用10毫升生长培养基替换。


        图2.转染后12-18小时的GFP对照板A.相位对比通道,10倍放大。比例尺表示400微米。 B.GFP通道,10倍放大。比例尺表示400微米。

    3. 抗生素选择
      1. 转染后两天,将每个10-cm平板放入生长培养基中的4×10-cm平板中以避免选择期间过度拥挤细胞(在分裂期间不要使用选择抗生素)。
      2. 细胞粘附在平板上后,取出培养基并用含有200μg/ ml潮霉素B的新鲜生长培养基替换。每3-4天更换一次新鲜潮霉素培养基。选择应该需要大约7-10天。通常预计每盘6-20个菌落(图3)。
        注意:进行杀伤曲线以确定200μg/ ml潮霉素B对于NIH / 3T3 Flp-In细胞是最佳的。根据潮霉素和细胞系的来源,最佳选择浓度可能略有不同。


        图3.潮霉素B选择后7天的代表性菌落。相差4倍放大。比例尺表示1,000μm。

      3. 细胞可以从1或2个10-cm平板汇集到6孔平板的单个孔中,并在没有选择抗生素的生长培养基中传代。
        注:此步骤仅针对报告构建体执行。丢弃不应产生菌落的GFP对照板。

  2. Wnt5a刺激分析
    实验设计:对于基本的Wnt5a刺激,包括一个刺激条件(+ Wnt5a,其中添加含Wnt5a的培养基)和一个对照条件(-Wnt5a,其中添加了含有控制缓冲液的培养基)用于每个报道细胞系。实验设置将根据您的测定应用而变化;有关其他类型刺激的详细信息,请参阅数据分析部分。
    1. 种子报告细胞每孔0.09百万/孔在聚-D-赖氨酸包被的48孔板中,每孔400μl生长培养基。细胞应该约90%汇合。
      备注:
      1. 通过向48孔板的每个孔中加入200μl聚-D-赖氨酸溶液(0.1mg / ml的水溶液;无菌过滤),在室温下孵育15分钟,除去聚D-赖氨酸溶液,并用400μl水洗涤孔三次。在电镀单元之前将板完全空气干燥(取下盖子)。涂层板也可以在室温下储存以备将来使用。
      2. 为了进行定量,我们通常将细胞置于每个实验条件的一式三份孔中。
    2. 第二天,轻轻移除培养基,并用含有10nM Wnt-C59的400μl生长培养基替换。 Wnt-C59抑制内源Wnt的加工和分泌。允许细胞在含有Wnt-C59的培养基中达到100%融合(通常为一天)。在Wnt5a刺激当天细胞应尽可能融合。
      注:如果单层细胞收缩或剥落,重复电镀。收缩的细胞信号不好。
    3. 用Wnt5a刺激细胞,轻轻除去培养基,并用含有10nM Wnt-C59和各自浓度的Wnt5a的培养基代替。对于模拟刺激,使用含有Wnt-C59和Wnt控制缓冲液的培养基。如果其他药物与Wnt5a一起使用,在加入Wnt5a-和含药物培养基之前,可能需要对药物进行预处理(通常为1小时)。
      在媒体更换期间避免干扰细胞单层
    4. 用Wnt5a在37°C孵育细胞6小时。
    5. 为了收获细胞用于流式细胞术分析,在37℃下每孔用100μl胰蛋白酶解离细胞3-5分钟。用500μl生长培养基中和胰蛋白酶,并将细胞悬浮液转移到1.5ml微量离心管中。
    6. 在4℃下将细胞以12,000×g克离心3分钟以沉淀细胞。
    7. 从每个样品中取出上清液。避免干扰颗粒。
    8. 在室温下用100-150μl流式细胞仪缓冲液重悬沉淀。通过移液混合直到样品均匀重新悬浮并通过滤网帽将细胞悬浮液压入圆底管中。
    9. 使用流式细胞仪分析细胞。我们经常使用Becton Dickinson FACScan并分析每个样品30,000个细胞。
    10. 用软件分析数据文件(,例如,FlowJo)。详情请参阅下一节。

数据分析

  1. 对于一般数据分析,通过流式细胞计数软件中的侧向散射和前向散射参数来关闭活细胞群以排除死细胞。野生型NIH / 3T3 Flp-In亲本细胞系(即,未转染的)用作自体荧光的参考;然而,我们通常不会根据GFP信号关闭细胞群,以确保整个活细胞群都包含在记者分析中。生成活门控群体的GFP荧光对细胞计数的原始直方图。覆盖来自每个待比较样品的直方图以获得每个样品群体之间的中值荧光差异(图4)。中位数的这种差异表示为百分比:[(对照中位数 - 刺激中位数)/对照中位数]×100(在图5B中标记为'%下调')。多个直方图可能会被叠加以进行比较或参考(图5A,图7A)。


    图4.使用WRK报告基因分析的基本分析。来自一组样品的叠加直方图显示在Wnt5a刺激(0.2μg/ ml Wnt5a)6小时后WRK报告细胞系中GFP-Kif26b荧光的下调。

  2. 对于剂量反应分析,我们分析了不同浓度的Wnt5a配体或小分子抑制剂(包括0剂量点)的最少六个样品。可以将中位数与浓度作图以产生剂量 - 反应曲线(图5B)。对于抑制剂,我们通常在固定浓度的Wnt5a存在下改变药物浓度以确定剂量 - 反应关系。


    图5.使用WRK报告基因测定的剂量 - 反应分析的实例原始直方图(A)和由此产生的剂量 - 反应曲线(B)显示GFP-Kif26b下调作为Wnt5a浓度WRK记者测定。

  3. 对于时间进程的实验,如图6所示的Kif26b稳定性分析,我们以固定的时间间隔用Wnt5a刺激样品,直到实验结束时,所有样品一次收获。
    中位数是针对刺激持续时间绘制的

    图6.使用WRK报告基因分析的时间过程实验的例子。如在WRK报道分子测定中所测量的,在不存在或存在Wnt5a刺激的情况下GFP-Kif26b周转的动力学。使用放线菌酮来阻断报告细胞中的新蛋白质合成。

  4. 对于定量期间的统计分析,我们使用最少三个生物学重复(铺板并用Wnt5a和/或抑制剂在同时培养物中处理的细胞)。为了评估两组数据之间的差异,我们执行双尾不成对的Student's t - 测试(图7B)。我们为每组代表平均值的标准误差的复制品包含了误差条,我们通过计算复制品中位数的标准偏差并将该数除以 N 的平方根产生,其中 N 是重复次数(图6,图7B)。


    图7.使用WRK报告基因分析的途径分析实验的例子。 在通过慢病毒转导的异位Shisa2表达后部分阻断Wnt5a诱导的报道分子活性。 A.代表性的覆盖直方图显示异位Shisa2表达对WRK报道系中Wnt5a诱导的GFP-Kif26b下调的影响。 Shisa2是Wnt受体的卷曲家族的拮抗剂(Yamamoto等人,2005)。包括Wnt5a或对照缓冲液处理对WRK报道基因系的影响作为参考。 B.量化图(A)所示的结果。对于以下比较进行T检验:对照病毒对无病毒,P <= em = 0.0957(不显着);控制病毒对比Shisa2病毒, 0.001(显着)。

笔记

通过该测定检测到的Wnt5a信号传导似乎对细胞密度高度敏感。当细胞尽可能融合时,信号活动发生得最好,当细胞少于100%汇合时,活性急剧下降。可能需要一些优化来确定特定细胞类型和应用的最佳电镀条件。

食谱

  1. 生长培养基
    DMEM补充:
    10%FBS
    1%谷氨酰胺
    1%青霉素 - 链霉素
  2. Wnt控制缓冲区
    1x PBS补充:
    0.1%牛血清白蛋白
    0.5%(w / v)CHAPS
  3. 用于流式细胞术的细胞重悬缓冲液
    补充有0.5%FBS的1x PBS

致谢

该协议摘自以下文章:Susman et。,2017。我们感谢Michael Greenberg(哈佛医学院)的讨论和支持。我们感谢加州大学戴维斯分校癌症中心流式细胞术核心(由P30 CA093373支持)的Bridgette McLaughlin提供仪器,培训和支持。 WRK报道分子检测的发展和表征得到美国癌症协会授予IRG-95-125-13和国立卫生研究院授予1R35GM119574-01授予H.H.Ho,T32GM007753授予M.W.Susman。作者宣称没有利益冲突。

参考

  1. Fuerer,C.和Nusse,R.(2010)。 用于探测和操作Wnt信号通路的慢病毒载体 PLoS One < 5(2):e9370。
  2. Ho,H.Y.,Susman,M.W.,Bikoff,J.B.,Ryu,Y.K。,Jonas,A.M.,Hu,L.,Kuruvilla,R.and Greenberg,M.E。(2012)。 Wnt5a-Ror-Disheveled信号传导构成了控制组织形态发生的核心发育途径。 < Proc Natl Acad Sci USA 109(11):4044-4051。
  3. Korinek,V.,Barker,N.,Morin,P.J.,van Wichen,D.,de Weger,R.,Kinzler,K. W.,Vogelstein,B。和Clevers,H。(1997)。 β-catenin-Tcf复合物在APC中的组成性转录激活 - / - 结肠癌。科学 275(5307):1784-1787。
  4. Nishita,M.,Itsukushima,S.,Nomachi,A.,Endo,M.,Wang,Z.,Inaba,D.,Qiao,S.,Takada,S.,Kikuchi,A。和Minami,Y。( 2010)。 Ror2 / Frizzled复合物通过调节蓬乱聚合来介导Wnt5a诱导的AP-1活化。分子细胞生物学 30(14):3610-3619。
  5. Ohkawara,B。和Niehrs,C。(2011)。 一种基于ATF2的萤光素酶报告基因,用于监测非爪蟾中的非规范Wnt信号>胚胎。 Dev Dyn 240(1):188-194。
  6. Susman,MW,Karuna,EP,Kunz,RC,Gujral,TS,Cantu,AV,Choi,SS,Jong,BY,Okada,K.,Scales,MK,Hum,J.,Hu,LS,Kirschner,MW, Nishinakamura,R.,Yamada,S.,Laird,DJ,Jao,LE,Gygi,SP,Greenberg,ME和Ho,HH(2017)。 驱动蛋白超家族蛋白Kif26b将Wnt5a-Ror信号连接到脊椎动物细胞和组织行为的控制。 / a> Elife 6。
  7. Veeman,M.T.,Axelrod,J.D。和Moon,R.T。(2003)。 第二个正典。不依赖β-catenin的Wnt信号传导的功能和机制。 Dev Cell 5(3):367-377。
  8. Yamamoto,A.,Nagano,T.,Takeara,S.,Hibi,M。和Aizawa,S。(2005)。 Shisa通过抑制骶管因子Wnt和FGF的受体蛋白质成熟来促进头部形成。 / a> Cell 120(2):223-35。
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Copyright Karuna et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Karuna, E. P., Susman, M. W. and Ho, H. H. (2018). Quantitative Live-cell Reporter Assay for Noncanonical Wnt Activity. Bio-protocol 8(6): e2762. DOI: 10.21769/BioProtoc.2762.
  2. Susman, M. W., Karuna, E. P., Kunz, R. C., Gujral, T. S., Cantu, A. V., Choi, S. S., Jong, B. Y., Okada, K., Scales, M. K., Hum, J., Hu, L. S., Kirschner, M. W., Nishinakamura, R., Yamada, S., Laird, D. J., Jao, L. E., Gygi, S. P., Greenberg, M. E. and Ho, H. H. (2017). Kinesin superfamily protein Kif26b links Wnt5a-Ror signaling to the control of cell and tissue behaviors in vertebrates. Elife 6.
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